A) NEUREGULIN/ERB-B SIGNALING REGULATES NEURONAL PLASTICITY: POSSIBLE RELEVANCE TO SCHIZOPRENIA[unreadable] [unreadable] 1. Isolation and Characterization of the Neuregulin-1 Type IV Isoform: Accumulating evidence supports the involvement of NRG-1 and ErbB-4 receptors in the etiology of schizophrenia. The NRG-1 gene generates numerous transcripts by using different transcriptional promoters and alternative splicing. Interestingly, a single nucleotide polymorphism (SNP8NRG243177) is located close to a region proposed to function as a promoter for the novel NRG-1 isoform denoted as Type IV. The SNP8NRG243177 T/T polymorphism is associated with higher levels of Type IV transcripts in postmortem tissue. Moreover, SNP8NRG243177 T/T, which maps within the previously identified schizophrenia at-risk haplotype, is associated with lower prefrontal activation and the development of psychotic symptoms. NRG-1 Type IV transcripts were originally identified by RT-PCR as partial RNA fragments. Therefore, it currently is not known whether these partial transcripts originated from full-length NRG-1 mRNAs, and whether these mRNAs encode for pro-NRG-1 proteins that are post-translationally processed to produce a biologically active form of NRG-1. Toward understanding a possible role of Type IV NRG-1 in the human brain, we isolated two full-length mRNAs encoding Type IV proteins. We found that these transcripts are translated to generate pro-NRG-1 type IV protein that is post-translationally processed, released from cells, and has the capacity to activate ErbB receptors and its downstream signaling pathways. This study provides the first evidence for the existence of NRG-1 Type IV protein. Experiments are in progress to determine if, and how, expression of NRG-1 Type IV protein is altered in schizophrenia.[unreadable] [unreadable] 2. Depotentiation of LTP by NRG-1: Understanding NRG/ErbB-4 signaling in the brain is important to understand how the pathway is altered in schizophrenia. We found that NRG-1 reverses ('depotentiates') long-term potentiation (LTP) at hippocampal CA1 glutamatergic synapses in an activity-dependent fashion. Inhibitors that selectively target ErbB tyrosine kinases block NRG-1-dependent depotentiation, and increase LTP levels at synapses already potentiated. Using patch clamp and cell biological techniques, we demonstrated that NRG-1 depotentiates LTP by selectively reducing AMPA, but not NMDA, receptor currents. Live imaging of hippocampal neurons transfected with AMPA receptors fused to superecliptic green fluorescent protein (seGFP), a form of GFP that only fluoresces strongly when expressed on the cell surface, indicates that NRG-1 stimulates the internalization of surface seGluR1-containing AMPA receptors. Consistent with these and our earlier findings, others have shown that NRG-1 and ErbB receptor hypomorphic mice develop normally, but have a reduction in glutamate receptor levels and manifest behavioral deficits. This novel regulation of LTP by NRG-1 has important implications for the modulation of synaptic homeostasis at glutamatergic synapses, which can affect cognition, learning and memory, and for understanding molecular mechanisms that underlie complex disorders like schizophrenia. [unreadable] [unreadable] 3. ErbB-4 Surface Clustering by PSD-95 at Inhibitory Hippocampal Neurons: To extend on our earlier work, showing that ErbB-4 directly interacts with the postsynaptic density protein PSD-95 at glutamatergic synapses, we investigated the developmental expression and trafficking the receptor. This interaction is of special interest because it may be altered in schizophrenia. Using immunofluorescence analysis in hippocampal slices and dissociated neurons in culture, we found that ErbB-4 receptors are expressed predominantly at glutamatergic synapses in GABAergic interneurons. The trafficking of ErbB-4 in cultured hippocampal neurons was investigated by surface protein biotinylation and antibody labeling of receptors in live cells. We found that ErbB-4 immunoreactivity in developing neurons precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating with, PSD-95-positive puncta. The surface fraction of dendritic ErbB-4 increases with age, and NRG-1 triggers its internalization in young and mature neurons. These findings enhance our understanding of the role of ErbB-4/PSD-95 protein interaction for NRG-mediated signaling at glutamatergic synapses.[unreadable] [unreadable] 4. Function of NMDA NR2C Receptor tested in cells from knockout mice: We previously reported that expression of the NR2C subunit of the NMDAR is regulated by NRG-1 in cultured organotypic slices from cerebellum. To study the function of the NR2C subunit, DNA sequences corresponding to the first 11 exons of the gene protein were removed by homologous recombination. In collaboration with the groups of Drs. Vicini and Wolfe, the NMDAR excitatory postsynaptic currents (EPSCs) were studied in solitary cerebellar neurons cultured in microislands from wild-type (WT) and NR2C-Bgal knock-in mice, as well as NR2A subunit knockout mice. Compared to WT cells, NR2C null granule neurons have larger NMDA-EPSCs, fast decaying currents and increased quantal content. The most striking result is a significant increase in the NMDA-EPSC peak amplitude and charge transfer in NR2C mutant cells which is mostly due to an increase in quantal size, as estimated from miniature NMDA-EPSCs. Interestingly, the protein levels of NR1, NR2A and NR2B in cerebella from 21-day-old NR2C mutant mice are decreased, as compared to wild-type, suggesting a possible compensatory response to the increased NMDA-EPSCs.[unreadable] [unreadable] B. ACTIVITY-DEPENDENT REGULATION OF MUSCLE TYPES[unreadable] [unreadable] 1. Role of activity in the adult: The effects of motor neuron electrical activity on TnI transcription were measured in vivo by imaging individual myofibers. The levels of transcription were measured in adult muscles transfected with the SURE and FIRE GFP reporter constructs before and after electrical stimulation. We found that slow, tonic depolarization up-regulated SURE transcription, while fast, phasic stimuli enhanced FIRE transcription. These results indicated that the TnI slow and fast enhancers sense, and respond to, distinct patterns of neuronal activity. [unreadable] [unreadable] 2.Identification of calcium-responsive factors: Next, we set out to identify the precise DNA elements and transcription factors that respond differentially to activity. Numerous lines of evidence indicate that the effects of activity are mediated by calcium released from the sarcoplasmic reticulum. NFAT and NFkB are two transcription factors that respond to calcium transients in T-cells to differentially regulate genes. We found that while NFAT represses transcription from FIRE in response to slow-patterned activity, NFkB increased FIRE transcription in response to fast-patterned stimuli. These experiments exemplify how muscles can modify their adult contractile properties in response to distinct types of exercise.[unreadable] [unreadable] 3. TEF3/Tead4 knockout mice: Tead4 (or TEF3) was another transcription factor analyzed for its potential role in regulating muscle development and regeneration. Tead4-floxed mice were generated by homologous recombination. Developmental studies indicated that ablation of the gene resulted in a preimplantation failure. Tead4-/- embryos do not to express trophectoderm specific genes, and the morulae do not produce trophoblast stem cells, trophectoderm, or blastocoel cavities, and therefore fail to implant. Future experiments with conditional Tead4 mutant mice will be needed to assess if Tead4 regulates fiber type properties during development.